Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. Chronic infection puts individuals at risk for the development of cirrhosis, hepatocellular carcinoma, and liver failure, making chronic hepatitis C the leading indication for liver transplantation. While HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional viral targets and combinations of antivirals will be needed for effective control. Functional studies on two HCV proteins, p7 and NS2, have been limited. Although both proteins are essential, neither is required for HCV RNA replication other than the NS2-3 protease-mediated cleavage to liberate the N-terminus of the NS3 serine protease/helicase. The ability to probe p7 and NS2 functions has changed dramatically with recent development of cell culture systems allowing both HCV RNA replication and virion production (HCVcc). Preliminary experiments utilizing monocistronic and bicistronic HCVcc genomes indicate that both p7 and NS2 are absolutely required for infectious virus production. NS2's role(s) in virus production is independent of its autoproteolytic activity;whether p7's role is related to its putative ion channel activity or other mechanisms remains to be determined. Using a combination of genetic, biochemical and structural approaches, we propose to dissect the roles of HCV p7 and NS2 in virus assembly and release. Genetic analyses will target conserved features including surface exposed residues on the NS2 autoprotease domain and residues postulated to be involved in its oligomerization, based on our recent x-ray structure. Intergenic chimeras, initially impaired for virus production, will be used to select and map second site suppressor mutations to yield a genetic map of p7 and NS2 interactions. These genetic studies will be complemented by biochemical and cell biological approaches to examine the effect of deleterious and compensating mutations on homo-oligomerization, subcellular localization, interactions with viral and cellular partners, and ion channel activity (p7). Finally, we propose to continue our structural studies that began with the post-cleavage form of the NS2 autoprotease (revealing a domain exchanged homodimer with two composite cysteine protease active sites) to include full-length NS2. Through these studies, we hope to begin to clarify the role of these understudied viral proteins in the HCV lifecycle and uncover additional avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is a leading cause of liver disease including cancer. This proposal aims to study the function of two understudied viral proteins that are essential for infectious virus production. The resulting findings should aid development of more effective treatments aimed at eradicating this deadly viral disease.